The overall goal of this program is to develop novel inhibitors of human protein acyltransferases (hPATs) that =re effective as cancer therapeutic agents. PATs represent new targets for anticancer drug development !because of their pivotal roles in regulating the subcellular localization of specific oncoproteins, especially certain Ras proteins. A major reason that inhibition of protein palmitoylation has not yet been therapeutically exploited is that hPATs have yet to be molecularly and biochemicaily characterized. Based on sequence and structural homologies with recently identified PATs in Saccharomyces cerevisiae, we selected a human protein called HIP3 for further study. Utilizing a number of techniques including: an in vitro PAT assay; siRNA abrogation of HIP3 expression; expression in yeast; and overexpression in mouse fibroblasts, we have found that HIP3 has PAT activity toward the farnesyl-dependent palmitoylation motif found in H-Ras. Interestingly, HIP3-trasfected fibroblasts form colonies, demonstrate anchorage-independent growth in soft agar, and form tumors in mice. In the continuation period of this project, these findings and our previously published work on hPATs will be used to further characterize the cellular functions of HIP3 and other putative hPATs, to lay the foundation for the development of small molecule inhibitors of hPATs to be evaluated as anticancer therapeutics. This will include: characterization of the PAT activity of HIP3 and other putative hPATs using biochemical and molecular approaches; examination of the substrate specificities of hPATs in intact cells; determination of the roles of hPATs in cell signaling, transformation, proliferation and apoptosis; and examination of combined antitumor effects of PAT inhibitors with other signaling inhibitors or anticancer drugs. These studies should confirm our identification of HIP3 as the first oncogenic human PAT, provide insight into the biological roles of HIP3 and other hPATs, and validate HIP3 as a target for the development }f anticancer drugs.